The rôle of the tapetum in the formation of sporopollenin-containing structures during microsporogenesis in Pinus banksiana

Summary In the microsporangium of Pinus the outer layer of the peritapetal membrane and the pro-orbicular cores are not only formed in a similar manner, but are composed of apparently identical materials. Precursors for this lipoidal material are produced by the tapetal protoplasts, as are the precursors of sporopollenin. Production the precursors is sequential and appears to involve different cytoplasmic structures.The sporopollenin synthesised by the tapetum condenses upon the pro-orbicular cores, the peritapetal membrane, the exine initials and, on fragmentation of the tapetum, parts of the disintegrating cytoplasm. The evident unpolarised nature of the tapetal protoplasts, and the sequential nature of the synthesis of the lipoid and the sporopollenin by them, may point to orbicule formation in gymnosperms being a necessary by-product of the development of the peritapetal membrane.     

Development and Histochemical Observations of Tapetum and Peritapetal Membrane in Anther of Pulsatilla chinensis

Tapetum of Pulsatilla chinensis is of secretory type. Its development proceeds rapidly in following sequence: (1) The stage of initiation-differentiation. At this stage cytological and histochemical features have been described in detail in this paper. (2) The stage of growth- synthesis: This stage appears to be the most important anabolic phase during the development of the tapetum. The salient features are that the tapetal cells become relatively enlarged and form two polyploid nuclei or aberrent polyploid nuclei resulting in synthetizing maximum proteins, fluorescing substances and maximum fluorescent Pro-Ubisch bodies in the tapetal cytoplasm. (3) The stage of secretion-disorganization: After the disintegration of the tapetal wall the enlarged naked cells appear at once. This is an important secretion period in which Pro-Ubisch bodies as well as all other fluorescing substances, carbohydrate or some enzymes are released into anther loculus. The naked cell layer becomes disorgnized until the beginning divition of the pollen grains into two ceils. As to peritapetal membrane of P. chinensis, mainly based on the membrane being on the outer side of the tapetum enclosing both the pollen, tapetal cytoplasm and Ubisch bodies, and the cellular configurations facing the pollen, Authors postulate that peritapetal membrane might be survival of the cytoplasmic membrane of tapetal cells. However, the peritapetal membrane of P. chinensis is similar to that of plasmodial, tapetum reported in certain Compositae and that of secretory tapetum reported in Pinus banksiana. Heslop-Harrison and Gupta et al. had conceded that the tapetal and peritapetal membrane belong to the general class of sporopollenin. On the contrary in P. chinensis the sporopollenin property of peritapetal membrane is only confined to its inner surface. But the thin mem- brane itself with the reticulate sporopollenin attched on its inner side appears negative staining reactions for sporopollenin though it has an ability to resist the acetolysis as well. In P. chinensis the Ubisch body is short necked flask shaped and their size is very similar. Ubisch body is either single or 2–5 in a group, resulting in compound bodies. When the Pro-Ubisch body is still within the tapetal cell it shows positive fluorescent reaction, while it eomletely unstains with Teluidine blue O. So Authors infer that the sporopollenin precur- sors may have permeated through Pro-Ubisch bodies. Finally, How sporopollenin precursor is synthesized in the tapetal cells, transported to pollen locula and polymerized into the sporopollenin on pollen, Ubisch body and peritapetal membrane? Future works along these problems may yield fruitful results.     

云南松小孢子发生过程中绒毡层细胞的超微结构研究

云南松(Pinus yunnanensis Fr.)在小孢子囊发育早期,绒毡层原生质体发生收缩,并伴随着细胞壁厚度的增加。脂肪微滴和孢粉素物质沉积而形成周缘绒毡层膜。随着孢粉素物质的产生,绒毡层细胞质明显地液泡化。孢粉素物质在绒毡层细胞膨大的内质网槽库中形成,随后被排放到近邻的小液泡内或游离于细胞质中。孢粉素物质也可在特化的含片层的质体中形成,孢粉素依附在片层膜上,或释放到细胞质中。     

Development of the Tapetum in Pinus banksiana Preceding Sporogenesis

Early in sporangial ontogeny, the cells destined to become thesporogenous and tapetal tissue differentiate in a strikinglysimilar manner. The first conspicuous step in development isa contraction of the protoplasts, beginning at the centre ofthe microsporangium and moving radially to its periphery. Similardevelopment of the two groups of cells ceases as the callosewall is formed around the meiocytes. At this point the originalwalls investing the tapetal cells become gelatinous, and lipidsynthesis commences within the contracted protoplasts. The bulkof this lipid is secreted from the cells, and becomes lodgedin the loculus, either as globules in the expanded radial andinner cell walls, or as a continuous layer on the inside ofthe middle lamella separating the loculus from the wall of themicrosporangium. This lipoidal layer forms the basement of aperitapetal membrane, believed to serve as a container for thefluid in which the young sporogenous cells are immersed. Examination of protein levels and ribosome numbers in the tapetalcells reveals that protein synthesis proceeds at an increasingrate throughout the development preceding meiosis, but apparentlyceases as the pollen mother cells become enveloped in callose.     

Tapetal Organization During Sporogenesis in Psilotum nudum

Stages in the differentiation of the tapetum of Psilotum nudumare described. Two concurrently occurring components of thetapetum can be recognized. A plasmodial tapetum with associatedfunctional nuclei develops within the sporangial loculus duringthe early stages of differentiation, appears to remain viablefor several months, that is during the entire period of sporogenesis,and undergoes reorganization on three occasions. During MeiosisI groups of spore mother cells are enclosed in clear areas withinthe plasmodium: by the end of Meiosis II each tetrad is isolatedin a plasmodial chamber; and, finally, mature spores are enclosedwithin individual tapetal chambers. Typically enlarged cellsare present during the development of a cellular, parietal tapetum.A sporopollenin-containing layer or tapetal membrane characteristicof a secretory tapetum develops on the inner tangential walland lines the surface of the loculus. This tapetal membranepersists even after dehiscence of the sporangium. These observationsare discussed in relation to previously published conflictingdata and may be relevant to the arguments concerning the relationshipof the Psilotaceae to the Filicales. Psilotum nudum, light microscopy, parietal tapetum, plasmodial tapetum, tapetal membrane, tapetal reorganization, sporogenesis, sporopollenin     

A histochemical and ultrastructural study of the ontogeny and differentiation of pollen inPodocarpus macrophyllus D. Don

Summary Male cones ofPodocarpus macrophyllus D. Don enter a period of dormancy lasting almost a year after the differentiation of archesporial tissue. The cell walls of the sporogenous and tapetal cells are different in composition from those of the cells comprising the wall of the microsporangium. The walls of tapetal cells undergo complete dissolution but the naked protoplasts do not invade the cavity of the microsporangium, and eventually degeneratein situ. Sporopollenin-containing bodies are formed on the tapetal plasmalemma although no specific tapetal organelles can be singled out as sites of synthesis of sporopollenin precursors. The original walls of the microspore mother cells are broken down completely and replaced by a thin callose-like wall. No cytomictic channels are formed prior to or during early meiosis. The outer nuclear membrane of the sporogenous cells forms numerous vesicles which likely play an important role in preparing the cell for meiosis and in the breakdown of the original sporogenous cell wall and the formation of the new wall. Pronounced evaginations and invaginations of the nuclear envelope during the tetrad stage are seen which again indicate vital nucleo-cytoplasmic exchange at the time when species specific sexine layer is being laid down. The microspore protoplast synthesizes a portion of sporopollenin precursors. Sexine and part of nexine I are laid down during the tetrad stage on lamellae of unit membrane dimensions while nexines II and III are formed after the dissolution of the tetrads by the coalescence of small, electron dense particles. Cells of the male gametophyte are initially separated from each other by distinct cell walls often traversed by plasmodesmata. Mature pollen grains have appreciable reserves of protein, lipid and starch. Results of histochemical and scanning electron microscopical observations are also reported and discussed.     

A Tapetal Membrane in Psilotum nudum (L.) Beauv

Ultrastructural studies (both SEM and TEM) on Psilotum nudumreveal that a tapetal membrane develops on the loculus sideof the inner tangential wall of a cellular, parietal tapetumwhich invests each sporangium within a synangium. The membraneconsists of orbicular projections of a homogeneous nature, supportedon a lamellated base; the whole structure is resistant to acetolysisand persists even at the dehiscence of the synangium. Pro-orbiculeswere not identified and the orbicles were not released intothe sporangial loculus. Spheroids, approx. 3 µm in diameter,are found in membrane-enclosed chambers within the plasmodialtapetum. Their formation is described as well as their finalstructure which reflects the structure of the mature sporodermfrom the outer exosporal layer outwards. Differentially stainingbodies occurring in the parietal tapetum and in the adjacentcells of the sporangium wall are implicated in the formationof the tapetal membrane. The bodies occur at the time when thesporangium wall is yellow and when plastids within the cellsof the sporangium wall develop large numbers of osmiophilicplastoglobuli. parietal tapetum, plasmodial tapetum, Psilotum nudum, spheroids, sporopollenin, tapetal membrane     

Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

POLLEN WALL AND TAPETAL ORBICULAR WALL DEVELOPMENT IN SORGHUM BICOLOR (GRAMINEAE)

Pollen wall development in Sorghum bicolor is morphologically and temporally paralleled by the formation of a prominent orbicular wall on the inner tangential surface of the tapetum. In the late tetrad stage, a thin, nearly uniform primexine forms around each microspore (except at the pore site) beneath the intact callose; concurrently, small spherical bodies (pro-orbicules) appear between the undulate tapetal plasmalemma and the disappearing tapetal primary wall. Within the primexine, differentially staining loci appear, which only develop into young bacula as the callose disappears. Thus, microspore walls are devoid of a visible exine pattern when released from tetrads. Afterwards, sporopollenin accumulates simultaneously on the primexine and bacula, forming the exine, and on the pro-orbicules, forming orbicules. Channels develop in the tectum and nexine, and both layers thicken to complete the microspore exine. Channeled sporopollenin also accumulates on the orbicules. A prominent sporopollenin reticulum interconnects the individual orbicules to produce an orbicular wall; this wall persists even after the tapetal protoplasts degenerate and after anthesis. While the pollen grains become engorged with reserves, a thick intine, containing conspicuous cytoplasmic channels, forms beneath the exine. Fibrous material collects beneath the orbicular wall. The parallel development and morphological similarities between the tapetal and pollen walls are discussed.     

Development and cell surface of a non-syncytial invasive tapetum inCanna: Ultrastructural,freeze-substitution,cytochemical and immunofluorescence study

Summary The anther ofCanna indica L. ×C. sp. hybrid contains a hitherto uncharacterized non-syncytial, invasive category of tapetum. With the onset of prophase I the tapetal walls are dissolved and the released protoplasts migrate into the loculus, where they stay discrete. Concomitant with the dissolution of walls the tapetal protoplasts develop a 17 nm thick extracellular granulo-fibrillar cell coat. This feature develops in the synchronous phase of tapetal development. The cell coat reacts positively with ruthenium red, potassium ferrocyanide, ConA-FITC and in the Thiéry reaction. Immunofluorescence microscopy using anti-tubulin revealed that even after the migration of tapetal cells into the loculus, the microtubules retain a predominant orientation in the cell cortex, probably derived from that in the original tapetal walled cells. This order is lost during late post-meiotic stages when the cells distort and can produce amoeboid processes. The microtubule orientation is correlated with that of the cell coat fibrils. Tapetal cells vary in ultrastructure and the density of cell coat fibrils after their migration into the loculus, but the cell coat persists until the cells degenerate. It is surmised that development of the cell coat relates to the lack of cell fusion and that the cortical microtubules help to sustain cell form. During post-meiotic stages the free tapetal cells develop massive peripheral arrays of interconnected ER cisternae, probably as part of a secretory apparatus which matures when the spores are producing their ornamented walls. Buds grown in colchicine solution showed accumulation of sporopolleninlike granules in all extracellular spaces of the anther cavity.     

An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.     

The tapetum inSchizaea pectinata (Schizaeaceae) and a comparison with the tapetum inPsilotum nudum (Psilotaceae)

A combination tapetum consisting of a cellular, parietal component and a plasmodial component occurs inSchizaea pectinata. A single, tapetal initial layer divides to form an outer parietal layer which maintains its cellular integrity until late in spore wall development. The inner tapetal layer differentiates into a plasmodium which disappears after the outer exospore has developed. In the final stages of spore wall development, granular material occurs in large masses and is dispersed as small granules throughout the sporangial loculus. No tapetal membrane develops. Comparisons are drawn with the combination tapetum found inPsilotum nudum.     

Colchicine inhibits plasmodium formation and disrupts pathways of sporopollenin secretion in the anther tapetum ofTradescantia virginiana L.

Summary During an earlier investigation, microtubules were observed at the periphery of invasion processes in the developing syncytial tapetum ofTradescantia virginiana L. They were also associated with membranous sacs that accumulate adjacent to tetrads, with putative fusion sites where the tapetal plasmodium is initiated, and, in postmeiotic stages, with the perispore membrane that encloses the developing spore cells. Colchicine was administered to developing flower buds to investigate the roles of these microtubules. The results indicate that microtubules neither initiate nor guide the tapetal invasion of the loculus. The treatments, however, resulted in absence of cell coat from invasion processes and prevention of cell fusion. They also inhibited polarized migration of membrane sacs and removed the associated microtubules. The development of an organized secretory apparatus at the perispore membrane was disrupted, with subsequent disordered deposition of sporopollenin in the extracellular spaces of the partially-fused plasmodium. The results suggest that microtubules participate in the formation and internal spatial organization of the tapetal plasmodium, and establishment of a secretory surface that normally produces sporopollenin at the tapetum-microspore interface.     

Microsporogenesis in Taxus baccata L.: The Formation of the Tetrad and Development of the Microspores

Following meiosis II in Taxus microsporangia a small proportionof the tetrads regularly degenerated. Despite frequent inequalityin the frequency of ribosomes between the spores of a tetrad,partial degeneration within a tetrad was never observed. Theinitial wall of the young spores was found to resemble the wallof the mother cell in containing a fibrillar layer, and thetwo walls may possess similar isolating properties. The symmetryof the tetrad was regularly iso-bilateral. The formation ofthe sporoderm began as the spores were released into the loculusby the rapid dissolution of the wall of the mother cell. Osmiophilicdroplets emerged from the spore protoplast and entered the wall.The fibrillar layer ceased to be recognizable and the dropletscoalesced to form an outer layer on which up to six sporopolleninlamellae, probably of tapetal origin, were deposited. The accretionof a single layer of sporopollenin droplets, in no recognizablepattern, gave rise to the outer verrucose part of the exine.Cytochemical tests showed that the tapetum was rich in acidphosphatases from the beginning of meiosis. Towards the endof its degeneration the tapetum intruded into the loculus andcould therefore be regarded as partly invasive. Taxus baccata, microsporogenesis, tetrad symmetry, sporoderm     

The Ultrastructural Aspect of Tapetum and Ubisch Bodies in Anemarrhena asphodeloides

From ontogeny of tapetum in Anemarrhena asphodeloides, the ultrastructnral features of tapetal cells are as follows: 1. The profuse rough endoplasmic reticula are often closely associated with lipid bodies and vesicles, and linking each other into compound organelles. This is one of the striking features in Anemarrhena tapetal cell. 2. After meiosis of the micro- spore mother cell, the tapetal cytoplasm contains a large number of vesicles, in which the electron opaque substances are accumulated. Then they fuse to form a large zone of storage material similar to lipid bodies. Before accumulation of opaque material, these vesicles in the tapetal cytoplasm are larger than those in elaioplast (see Plate II, Fig. 2). 3. During stage of pollen maturation the tapetal cytoplasm becomes disorganized and the cells are almost occupied by the elaioplasts at various degree of development. On the basis of the report of Dickinson (1973), the formation of a pollen coatings of Lilium is different from that of Raphanus. The osmiophilic bodies in the former have originated from membrane lamellae or membranous system of plastid, and those in the latter are formed from the plastid vescles. It is intereting to note that the mode of origin of the plastid osmiophilic bodies in Anemarrhena is rather similar to that of Raphanus than to Lilium. About the origin of the pro-Ubisch bodies in tapetal cytoplasm of Anemarrhena studies revealed that a large number of the medium electron dense bodies appear in the tapetal cytoplasm. This is the first sign of the formation of the pro-Ubisch bodies and its character is very similar to spherosome in many respects. From many ultrasections, it can be seen that the ER profile is closely associated with the pro-Ubisch bodies. Thus we can conclude that the proubisch bodies of Anemarrhena are derived from rough endoplasmic reticulum. Although Heslop-Harrison et al. (1969) has considered that the compound Ubisch bodies do not occur in Lilium, there are prominent aggregation of Ubisch bodies in Anemarrhena, same as reported in Oxalis (Cariel, 1967), Silene (Heslop-Harrison, 1963a) and Helleborus (Echlin et al., 1968). After investigation on certain angiosperm in 1972, Gupta and Nanda have reported that the peritapetal membrane belonging to tapetum of secretory type lies against the inner tang- ential wall; in the plasmodial type of tapetum, it is formed on the outer tangential wall. But in some species of Poaceae and Solanaceae, the peritapetal membrane is formed on both sides of the tapetal cells (Banerjee, 1967; Reznickov & Willemse, 1980). In the secretory tapetum of Anemarrhena, the peritapetal membrane, which do not comply with the conclusion of Gupta & Nanta (1972), is formed on outer tangential wall.     

Pollen and Tapetum Development in Male Fertile Rosmarinus Officinalis L. (Lamiaceae)

The development of microspores/pollen grains and tapetum was studied in fertile Rosmarinus officinalis L. (Lamiaceae). Most parts of the cell walls of the secretory anther tapetum undergo modifications before and during meiosis: the inner tangential and radial cell walls, and often also the outer tangential and radial wall, acquire a fibrous appearance; these walls become later transformed into a thin poly-saccharidic film, which is finally dissolved after microspore mitosis. Electron opaque granules found within the fibrous/lamellated tapetal walls consist of sporopollenin-like material, but cannot be interpreted as Ubisch bodies. The middle lamella and the primary wall of the outer tangential and radial tapetal walls remain unmodified, but get covered by an electron opaque, sporopollenin-like layer. Pollenkitt is formed only by lipid droplets from the ground plasma and/or ER profiles, the plastids do not form pollenkitt precursor lipids. Tapetum maturation (“degeneration”) does not take place before late vacuolate stage.

The apertures are determined during meiosis by vesicles or membrane stacks on the surface of the plasma membrane. The procolumellae are conical, but at maturity the columellae are more cylindrical in shape. The columellar bases often fuse, but a genuine foot layer is lacking. The formation of the endexine starts with sporopollenin-accumulating white lines adjacent to the columellar bases. Later, the endexine grows more irregularly by the accumulation of sporopollenin globules. In mature pollen the intine is clearly bilayered.

Generative cells (GCs) and sperm cells contain a comparatively large amount of cytoplasm, and organelles like mitochondria, dictyosomes, ER, and multi-vesicular bodies, but no plastids; GCs and sperms are separated from the vegetative cell only by two plasma membranes.     

The loculus content and tapetum during pollen development in Lilium

 The ratio of loculus volume to the volume of the entire anther began to increase from the microspore mother cell stage and reached 32.3% at anthesis. The content of the loculus was examined in Lilium during pollen development and two waves could be distinguished. From the premeiotic stage until the vacuolated microspore stage, the loculus consisted of neutral polysaccharides, pectins and proteins. These substances originated from tapetal activity from the premeiotic stage until the young microspore stage. Dictyosomes and rough endoplasmic reticulum seemed to be involved in tapetal secretion, although, in some mitochondria, vesicles progressively developed as early as premeiosis and increased until the young microspore stage, which could reveal their involvement in the secretion process. At this stage, numerous cytoplasmic vesticles containing material similar to the locular material fused with the plasma membrane of the tapetum so that vesicle content was in contact with the loculus. It seems that tapetal and callose wall degradation at the late tetrad stage may also have contributed to the production of material in the loculus. From pollen mitosis to anthesis, the anther loculus contained mainly the pollenkitt which was synthesized in the tapetum between the young microspore stage and the vacuolated microspore stage. At the young microspore stage, proplastids divided and developed into elaioplasts and smooth endoplasmic reticulum (SER) increased dramatically. Pollenkitt had a double origin: some droplets were extruded directly from the plastid stroma through the plastid envelopes; the others were unsaturated lipid globules, which presumably derived from the interaction between SER saccules and plastids. Received: 2 September 1997 / Revision accepted: 12 March 1998